Droplet based microfluidics holds great potential for high throughput screening applications. The encapsulation of single cells into droplets allows screening of cell products such as antibodies at very high throughput, e.g. up to several hundred thousand samples per day. For example, individual antibody-secreting cells (e.g. hybridoma- or B-cells) can be encapsulated into droplets together with a recombinant drug target of interest, such as an enzyme. Addition of a fluorogenic substrate of the enzyme will then reveal which droplets contain a cell releasing antibodies which inhibit enzymatic activity and facilitate the specific sorting of these. However, for many screening applications, it would be very useful to co-encapsulate (in addition to the antibody secreting cell) another reporter cell, rather than a recombinant enzyme. This reporter cell could mediate a fluorescence signal (e.g. expression of a reporter enzyme which can easily be detected in droplets; such as (3-Gal) upon the desired effect of an antibody, such as the stimulation or inhibition of cellular pathways upon antibody binding of G protein-coupled receptors (GPCRs). GPCRs are the targets of most best-selling drugs and about 40% of all prescription pharmaceuticals.
A crucial step for this kind of screens is the generation of droplets hosting both cell types, optionally at the single cell level. Non-deterministic cell encapsulation is limited by Poisson statistics, meaning that the number of cells per droplets varies significantly. Alternative deterministic encapsulation procedures have been described, however, until today the reliable co-encapsulation of two different cell types has not been demonstrated, especially not at single-cell level. Therefore, cell-based assays requiring the co-encapsulation of two different cell types at the single cell level (as required for drug screening; e.g. one B-cell secreting antibodies and another reporter cell indicating the effect of the antibody on a cellular target) can hardly be performed in droplets. This is due to the fact that the cell occupancy in each droplet cannot be controlled tightly, including controlled co-encapsulation of cells and beads as required for antibody binding assays. Furthermore, the quantitative analysis of antibody binding is challenging, as the bead (and any fluorescently labelled objects bound to it) can float out of the focal plane and/or out of the centre of the laser spot (having the highest intensity).
To overcome these limitations, the inventors have developed novel sorting devices and sorting methods allowing for the selection of droplets for any desired droplet occupancy, including the presence of two different cell types such as an antibody secreting cell (or any other “effector cell”) and a reporter cell. Furthermore, they have developed a system, more specifically a particular labelling approach of binding partners in droplets, which allows for quantitative binding assays, even if the object of interest is out of the focal plane or outside the most intense laser spot. Taken together, this should have manifold applications in drug screening and antibody discovery, a market with annual sales exceeding 50 billion USD.